苎麻资源核心种质构建方法及遗传多样性研究
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摘要
本研究从构建核心种质的技术方法入手,建立苎麻种质资源核心种质的构建程序,初步构建苎麻种质资源核心种质,并在分子水平对核心种质进行遗传多样性评价,为苎麻种质资源的进一步深入研究打下基础。
1、苎麻核心种质构建方法研究：利用国家种质长沙苎麻圃的790份苎麻种质资源,在已有的25个性状数据的基础上,采用不同聚类方法、不同抽样方法、不同遗传距离构建苎麻核心种质,用质量性状和数量性状的6个指标评价不同结合方式(聚类方法、抽样方法、遗传距离)构建核心种质的优劣。结果表明,不同的取样方法对质量性状和数量性状的遗传多样性影响不同,优先取样+多次聚类随机取样方法能使质量性状品系间的差异达到最大化,而优先取样+多次聚类变异度取样方法能使数量性状品系间的差异达到最大化。用优先取样+多次聚类随机取样方法取样时,采用最短距离法和重心法构建的核心种质最好,用优先取样+多次聚类变异度取样时,采用离差平方和法则是构建苎麻核心种质的最佳聚类方法。苎麻核心种质构建与质量性状的不同遗传距离无关,但数量性状以欧氏距离最佳。
2、初步构建苎麻种质资源核心种质：采取优先取样+多次聚类变异度取样方法,在20%的抽样水平下,构建了158份苎麻初级核心种质。构建的核心种质的质量性状多样性指数均值、数量性状方差差异百分率、变异系数变化率均高于原种质,极差符合率100%；质量性状的性状频率分布与原种质有差异的性状为12.5%；原种质各数量性状间的相关性基本上在核心种质中得到保留。
3、苎麻EST-SSR引物的开发及利用：SSR重复次数的变异引起位点长度的变化,是产生SSR多态性的主要原因。本试验设计的76对引物的SSR重复次数在3-44之间。重复4次所占比例最高,占所检测的SSR的30%；重复3次和重复18次次之,均占检测SSR的17.1%。重复基元长度变化是EST-SSR位点多态性的主要表现形式,苎麻EST-SSR重复基元长度变化区间在12-44之间,其中,重复基元长度主要为12bp和18bp,这两种基元长度占所检测SSR总数的68.4%。
4、苎麻核心种质遗传多样性分子评价：利用76对EST-SSR引物,对62份苎麻种质DNA进行扩增,其中70对引物进行了有效扩增,50对引物具有多态性,27对引物扩增出了清晰条带。利用筛选出的27对SSR引物对158份苎麻核心种质DNA进行PCR扩增。共扩增出64条多态性条带,多态性条带集中在80-200bp之间。平均每对引物扩增出2.37条条带。
对158份苎麻核心种质进行分子标记遗传多样性分析表明,其相似系数在0.27至0.90之间,平均相似系数0.61。通过聚类分析,可以把158份核心种质资源分为10类。采用19个田间性状,通过卡方距离和最短距离法对158份核心种质进行聚类分析,与分子标记的聚类结果进行比较,表明田间性状的聚类结果与分子标记的聚类结果有相似性。
The primary core collection of ramie (Boehmeria Jacq.)was established based on the studies of the technical method, the genetic diversity of the core collection was evaluated on the molecular level.
a. Studies on the method of core-collection establishment of ramie:Using different clustering methods, different sampling strategy and different genetic distances of qualitative and quantitative characters to establish core collection of ramie based on the data of25traits of790accessions from the Field Genebank for Ramie(Changsha).6Genetic parameters were used to evaluate the core collections established with different methods. The results showed that different sampling methods have different impacts on qualitative traits and quantitative traits. For the largest genetic diversity of qualitative traits, it is effective to choose stepwise clustering with random sampling strategy and preferred sampling strategy. For the largest genetic diversity of quantitative traits, it is effective to choose stepwise clustering with deviation sampling strategy and preferred sampling strategy. The ramie core collection is the best one constructed by centroid method, single linkage clustering way under stepwise clustering with random sampling strategy and preferred sampling strategy and constructed by ward's clustering way under stepwise clustering with deviation sampling strategy and preferred sampling strategy. Core collection in ramie was not related with genetic distances of qualitative traits, whereas the core collection of ramie constructed by euclidean distance of quantitative traits was the best.
b. Establishing of primary core collection of ramie:The primary core collection of ramie which contained158accessions was established using the method of stepwise clustering with deviation sampling strategy and preferred sampling strategy under20%sampling level. The index of genetic diversity, variance difference percentage and variable rate of coefficient of variation of the core collection were higher than the total collection. The coincidence difference percentage was100%.
c. Development of EST-SSR primer for ramie:Different SSR repeats causes changes of the site length which results in SSR Polymorphism. There were76EST_SSR primers designed in the experiment whose SSR repeats were from3to44. Most of them was4repeats (account for30%),3repeats and18repeats account for17.1%of SSRs, respectively. The polymorphic primers of SSR mainly derived from changes in the length of repeat motifs, which ranged froml2bp to44bp. There are mainly12bp and18bp for the length of repeat motifs, accounting for68.4%of EST-SSRs.
d. Evaluation of genetic diversity of the core collection by molecular markers:76primer-pairs were tested among62ramie DNA, the PCR products showed that70pairs were effectively amplified. Among the70pairs,50pairs obtained polymorphic PCR bands in which27pairs obtained clear polymorphic bands. The27pairs of SSR primers amplified64polymorphic bands with the average2.37per SSR primer pair in the158cultivars of the core collection. Most of the band varied from80to200bp.
SSR technique was used in analyzing the biological genetic diversity of158core collection, and the results showed:the average genetic similarity is0.61, varying from0.27to0.90. The158accessions were grouped into10types based on the cluster analysis. Similarity results were obtained by shortest distance method and chi-square distance based on19field traits.
1、苎麻核心种质构建方法研究：利用国家种质长沙苎麻圃的790份苎麻种质资源,在已有的25个性状数据的基础上,采用不同聚类方法、不同抽样方法、不同遗传距离构建苎麻核心种质,用质量性状和数量性状的6个指标评价不同结合方式(聚类方法、抽样方法、遗传距离)构建核心种质的优劣。结果表明,不同的取样方法对质量性状和数量性状的遗传多样性影响不同,优先取样+多次聚类随机取样方法能使质量性状品系间的差异达到最大化,而优先取样+多次聚类变异度取样方法能使数量性状品系间的差异达到最大化。用优先取样+多次聚类随机取样方法取样时,采用最短距离法和重心法构建的核心种质最好,用优先取样+多次聚类变异度取样时,采用离差平方和法则是构建苎麻核心种质的最佳聚类方法。苎麻核心种质构建与质量性状的不同遗传距离无关,但数量性状以欧氏距离最佳。
2、初步构建苎麻种质资源核心种质：采取优先取样+多次聚类变异度取样方法,在20%的抽样水平下,构建了158份苎麻初级核心种质。构建的核心种质的质量性状多样性指数均值、数量性状方差差异百分率、变异系数变化率均高于原种质,极差符合率100%；质量性状的性状频率分布与原种质有差异的性状为12.5%；原种质各数量性状间的相关性基本上在核心种质中得到保留。
3、苎麻EST-SSR引物的开发及利用：SSR重复次数的变异引起位点长度的变化,是产生SSR多态性的主要原因。本试验设计的76对引物的SSR重复次数在3-44之间。重复4次所占比例最高,占所检测的SSR的30%；重复3次和重复18次次之,均占检测SSR的17.1%。重复基元长度变化是EST-SSR位点多态性的主要表现形式,苎麻EST-SSR重复基元长度变化区间在12-44之间,其中,重复基元长度主要为12bp和18bp,这两种基元长度占所检测SSR总数的68.4%。
4、苎麻核心种质遗传多样性分子评价：利用76对EST-SSR引物,对62份苎麻种质DNA进行扩增,其中70对引物进行了有效扩增,50对引物具有多态性,27对引物扩增出了清晰条带。利用筛选出的27对SSR引物对158份苎麻核心种质DNA进行PCR扩增。共扩增出64条多态性条带,多态性条带集中在80-200bp之间。平均每对引物扩增出2.37条条带。
对158份苎麻核心种质进行分子标记遗传多样性分析表明,其相似系数在0.27至0.90之间,平均相似系数0.61。通过聚类分析,可以把158份核心种质资源分为10类。采用19个田间性状,通过卡方距离和最短距离法对158份核心种质进行聚类分析,与分子标记的聚类结果进行比较,表明田间性状的聚类结果与分子标记的聚类结果有相似性。
The primary core collection of ramie (Boehmeria Jacq.)was established based on the studies of the technical method, the genetic diversity of the core collection was evaluated on the molecular level.
a. Studies on the method of core-collection establishment of ramie:Using different clustering methods, different sampling strategy and different genetic distances of qualitative and quantitative characters to establish core collection of ramie based on the data of25traits of790accessions from the Field Genebank for Ramie(Changsha).6Genetic parameters were used to evaluate the core collections established with different methods. The results showed that different sampling methods have different impacts on qualitative traits and quantitative traits. For the largest genetic diversity of qualitative traits, it is effective to choose stepwise clustering with random sampling strategy and preferred sampling strategy. For the largest genetic diversity of quantitative traits, it is effective to choose stepwise clustering with deviation sampling strategy and preferred sampling strategy. The ramie core collection is the best one constructed by centroid method, single linkage clustering way under stepwise clustering with random sampling strategy and preferred sampling strategy and constructed by ward's clustering way under stepwise clustering with deviation sampling strategy and preferred sampling strategy. Core collection in ramie was not related with genetic distances of qualitative traits, whereas the core collection of ramie constructed by euclidean distance of quantitative traits was the best.
b. Establishing of primary core collection of ramie:The primary core collection of ramie which contained158accessions was established using the method of stepwise clustering with deviation sampling strategy and preferred sampling strategy under20%sampling level. The index of genetic diversity, variance difference percentage and variable rate of coefficient of variation of the core collection were higher than the total collection. The coincidence difference percentage was100%.
c. Development of EST-SSR primer for ramie:Different SSR repeats causes changes of the site length which results in SSR Polymorphism. There were76EST_SSR primers designed in the experiment whose SSR repeats were from3to44. Most of them was4repeats (account for30%),3repeats and18repeats account for17.1%of SSRs, respectively. The polymorphic primers of SSR mainly derived from changes in the length of repeat motifs, which ranged froml2bp to44bp. There are mainly12bp and18bp for the length of repeat motifs, accounting for68.4%of EST-SSRs.
d. Evaluation of genetic diversity of the core collection by molecular markers:76primer-pairs were tested among62ramie DNA, the PCR products showed that70pairs were effectively amplified. Among the70pairs,50pairs obtained polymorphic PCR bands in which27pairs obtained clear polymorphic bands. The27pairs of SSR primers amplified64polymorphic bands with the average2.37per SSR primer pair in the158cultivars of the core collection. Most of the band varied from80to200bp.
SSR technique was used in analyzing the biological genetic diversity of158core collection, and the results showed:the average genetic similarity is0.61, varying from0.27to0.90. The158accessions were grouped into10types based on the cluster analysis. Similarity results were obtained by shortest distance method and chi-square distance based on19field traits.
引文
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